Stroke is a major killer in the USA. The underlying pathophysiology for stroke is ischemia and reperfusion brain injury. Although many neuroprotective interventions have been identified, clinically practical methods to reduce ischemic brain injury are not well established yet. Our long-term research goals are to identify effective interventions for ischemic brain injury and to understand the mechanisms of these interventions. Our preliminary results suggest a novel neuroprotective method in which a prior brief exposure to isoflurane (preconditioning) is used to induce ischemic tolerance. Initial studies suggest that the mechanisms for this isoflurane preconditioning (IsoP)-induced neuroprotection involve alteration of glutamate transporter function in the acute phase and activation of p38 mitogen-activated protein kinase (MAPK) in the delayed phase. The proposed studies will: 1) investigate which glutamate transporters are involved and how the functional changes of the involved glutamate transporters translate into improved neurological outcome; 2) examine how p38 is activated by isoflurane; 3) determine whether the high mobility group (HMG) I (Y) transcription factor is downstream of p38 for IsoP-induced neuroprotection. Brain slices and in vivo temporary middle cerebral arterial occlusion models will be used. Neuronal survival will be evaluated by histological analysis. Extracellular glutamate levels in the ischemic penumbra will be quantified by microdialysis. Antisense oligonucleotides will be used to downregulate selective proteins such as glutamate transporters, p38 MAPK kinases and HMG I(Y). Western analysis and immunohistochemistry will be used to quantify and localize protein expression. Brain regional expression of HMG I(Y) will be determined by quantitative in vitro autoradiography. These studies will provide insight into the mechanisms of neuroprotection by IsoP and may elucidate mechanisms that could be exploited by new drugs. Since isoflurane has been used safely in clinical practice for many years, IsoP may prove to be a useful clinical method to reduce ischemic brain injury.